Epithelial-mesenchymal transition (EMT) is a transdifferentiation process by which epithelial cells undergo changes in morphology and cell-cell junction, to detach from each other and acquire invasive abilities. EMT is recognized to play key roles in processes such as wound healing, tissue fibrosis and cancer. With regard to cancer, EMT not only allows cancer cells to disseminate from the primary tumor but also confers protection from cell death, facilitates immune escape and induces resistance to therapy. TGF beta and Wnt signaling are considered the major drivers of EMT, however recent findings indicated that both of these pathways are regulated by a third one named Hippo. In fact, downstream mediators of this pathway namely YAP, TAZ and TEAD were found to act independently or as co-activators for both beta catenin/TCF and Smad complexes to induce EMT. Based on this, the development of approaches to inhibit activity of the Hippo pathway is expected to have applications for the treatment of EMT-associated diseases.
The adenosine monophosphate-activated protein kinase (AMPK) is an important regulatory protein for cellular energy balance and is considered a master switch of glucose and lipid metabolism in various organs, especially in skeletal muscle and liver. Recent evidence indicated that the function of this enzyme extends beyond the canonical metabolic pathway to include tumor suppression, cell polarity, fibrosis, and even aging. Concerning its role in metabolism, AMPK was originally defined as the upstream kinase for the critical metabolic enzymes Acetyl-CoA carboxylase (ACC1 & ACC2) and HMG-CoA reductase, which serve as the rate limiting steps for fatty-acid and sterol synthesis in a wide-variety of eukaryotes. In specialized tissues such as muscle and fat, AMPK regulates glucose uptake via the RabGAP TBC1D1, which along with its homolog TBC1D4 (AS160), play key roles in GLUT4 trafficking following exercise and insulin. In skeletal muscles, AMPK stimulates glucose transport and fatty acid oxidation, and in the liver, it augments fatty acid oxidation and decreases glucose output, cholesterol and triglyceride synthesis. These metabolic effects induced by AMPK are associated with lowering blood glucose levels in hyperglycemic individuals.
In conditions where nutrients are scarce, AMPK acts as a metabolic checkpoint inhibiting cellular growth. The most thoroughly described mechanism by which AMPK regulates cell growth is via suppression of the mammalian target of rapamycin complex 1 (mTORC1) pathway. This occurs by direct phosphorylation of the tumor suppressor TSC2 and also through direct phosphorylation of Raptor (regulatory associated protein of mTOR), on two conserved serines, which blocks the ability of the mTORC1 kinase complex to phosphorylate its substrates. mTORC1 has been shown to induce cell growth through inhibition of autophagy, a cellular process of “self engulfment” in which the cell breaks down its own organelles (macroautophagy) and cytosolic components (microautophagy) to ensure sufficient metabolites when nutrients run low. In addition to inhibitory phosphorylation of mTORC1, studies from a number of laboratories in the past few years have revealed that AMPK directly activates the ULK1, a kinase with a critical role in autophagy and mitochondrial homeostasis.
AMPK has also been shown to mediate the tumor suppressive function of the liver kinase LKB1, a gene associated with Peutz-Jeghers syndrome, an autosomal dominant genetic disorder characterized by multiple hamartomatous polyps (benign overgrowth of differentiated tissues) in the gastrointestinal tract and a markedly increased risk of gastrointestinal adenocarcinomas, of lung adenocarcinomas, 19% of squamous cell carcinomas and 20% of cervical carcinomas and other cancers
In addition to the well-established role for AMPK in cell growth and metabolism, recent studies suggested that AMPK may control cell polarity and cytoskeletal dynamics. In fact, it has been known for some time that the AMPK upstream effector, LKB1 plays a critical role in cell polarity from simpler to complex eukaryotes. These studies also supported a role for AMPK in cell polarity as a loss of this enzyme in Drosophila results in altered polarity and its activation in mammalian MDCK cells was needed for proper re-polarization and tight junction formation.
Loss of cell polarity is a consequence of epithelial mesenchymal transition (EMT). During this process epithelial cells lose their intercellular connections (tight junctions and adherens junction), change morphology and separate from each other. The affected cells generally adopt a spindly (elongated) morphology that facilitates their migration to distant sites. EMT has been shown to play key roles in development, cancer and organ fibrosis. AMPK has been shown to exert its inhibitory effect on renal fibrosis induced by TGF-β, angiotensin II, aldosterone, and high glucose, principally through inhibition of EMT. Moreover, the AMPK activator Metformin also suppresses EMT and thiazolidinediones were found to improve hepatic fibrosis by activating the AMPK signaling pathway in rats with non-alcoholic steatohepatitis. AMPK also plays a role in cardiac remodeling, as it pertains to diabetic cardiomyopathy, cardiac hypertrophy, and heart failure, suggesting that there might be therapeutic value in targeting the AMPK signaling pathway to treat cardiovascular diseases. In fact, dysfunction of the AMPK signaling pathway has been shown to be involved in the genesis and development of various cardiovascular diseases, including atherosclerosis, hypertension and stroke. AMP-activated protein kinase activator AICAR acutely lowers blood pressure and relaxes isolated resistance arteries of hypertensive rats. Adiponectin, a hormone AMPK activator has been shown to inhibit doxorubicin-induced cardiotoxicity.
In addition to its well established role in metabolism, cancer and fibrosis, AMPK has also been shown to affect other aging-associated diseases such as inflammation, neurodegeneration, sarcopenia and even the aging process itself. This is exemplified by the findings that the AMPK activator AICAR inhibits TNF-a- and IL-1a-induced NF-B reporter gene expression dose dependently in immune cells and inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) expression in stimulated macrophages. Activators of AMPK were also reported to inhibit chemotaxis in the monocyte-like cell line U937. In addition, AICAR profoundly inhibited lipopolysaccharide and IFN-α-stimulated production of the proinflammatory molecules nitric oxide synthase, COX-2, and IL-6. With regard to neurodegeneration, emerging studies indicate that AMPK signaling can regulate tau protein phosphorylation and amyloidogenesis, the major hallmarks of AD. AMPK is also a potent activator of autophagic degradation which seems to be suppressed in AD.
Sarcopenia is characterized by a muscle atrophy (a decrease in the size of the muscle), along with a reduction in muscle tissue “quality,” caused by such factors as replacement of muscle fibers with fat, an increase in fibrosis, changes in muscle metabolism, oxidative stress, and degeneration of the neuromuscular junction. Combined, these changes lead to progressive loss of muscle function and frailty. Agents that activate AMPK such as AICAR and GW501516 induced improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength.
Many studies with lower organisms have revealed that increased AMPK activity can extend the lifespan. Experiments in mammals have demonstrated that AMPK controls autophagy through mTOR and ULK1 signaling which augment the quality of cellular housekeeping. Moreover, AMPK-induced stimulation of FoxO/DAF-16, Nrf2/SKN-1, and SIRT1 signaling pathways and improves cellular stress resistance. Emerging studies indicate that the responsiveness of AMPK signaling clearly declines with aging. The loss of sensitivity of AMPK activation to cellular stress impairs metabolic regulation, increases oxidative stress and reduces autophagic clearance. These age-related changes activate immune cells, triggering a low-grade inflammation and metabolic disorders, leading to acceleration of aging. In contrast, evidence was provided that chronic feeding of rodents with AMPK agonists improves muscle endurance, reduces metabolic diseases, allows proper circadian regulation, and suppresses tumorigenesis. These findings strengthen AMPK's position as a main beacon of hope for the prevention and/or treatment of the current epidemic of metabolic and age-related diseases.
AMPK is generally activated in response to nutrient deprivation, exercise and also by hormones such as leptin, grelin, and adiponectin. Two classes of oral antihyperglycemic drugs (biguanidines and thiazolidinediones) have been shown to exert some of their therapeutic effects by directly or indirectly activating AMPK. Novel pharmacological agents such as the prototypical activator 5-aminoimidazole-4-carboxamide 1-D-ribonucleoside (AICAR) and Abbott A769662 have recently been introduced. Interestingly, oral administration of AICAR to eight-week-old male C57B/6J mice was reported to mediate a 44% increase in endurance without exercise in untrained mice, leading to speculation that these type of compounds may be considered as exercise mimetics. However, side effects and an acquired resistance to these drugs emphasize the need for the development of novel and efficacious AMPK activators.